A cell-based assay for IκBα stabilization using a two-color dual luciferase-based sensor
A cell-based assay for IκBα stabilization using a two-color dual luciferase-based sensor
复制标题
DOI：
10.1089/adt.2006.048
复制
发表时间：
2007-02-01
影响因子：
1.8
通讯作者：
Auld, Douglas S.
中科院分区：
文献类型：
--
作者：
Davis, R. Eric;Zhang, Ya-Qin;Auld, Douglas S.
在活化的 B 细胞样弥漫性大 B 细胞淋巴瘤细胞系 OCI-Ly3 中开发了一种用于稳定 I kappa B α 的细胞传感器测定法。该细胞系由于 I kappa B 激酶 (IKK) 的高组成活性而表达已知的核因子 kappa B (NF kappa B) 靶基因，IKK 会磷酸化蛋白质 I kappa B α，导致 I kappa B α 被蛋白酶体降解并激活 NF kappa B。细胞传感器测定使用绿光和红光发射甲虫荧光素酶，其中绿色荧光素酶与 I kappa B α 融合（I kappa B alpha-CBG68）和红色荧光素酶（CBR）以其天然状态存在。 I kappa B alpha-CBG68 报告基因充当 IKK 和蛋白酶体活性的传感器，而 CBR 用于使细胞数量和非特异性效应标准化。两种报告基因构建体均稳定整合，并置于诱导型启动子系统的控制下，当检测孵育与多西环素报告基因诱导同时进行时，该系统增加了对抑制剂的反应性倍数。该测定被小型化为 1,536 孔板格式，并显示 Z' 为 0.6；然后通过基于浓度响应的筛选策略，将其用于筛选 2,677 种生物活性化合物。然后使用 I kappa B α-CBG68 和 CBR 信号的浓度效应曲线来识别 I kappa B α 的特定稳定剂，例如 IKK 抑制剂或蛋白酶体抑制剂，它们会增加强力霉素诱导的 I kappa B α-CBG68 的升高，而不影响 CBR 的升高。从生物活性集合中鉴定出已知的和意想不到的 NF kappa B 信号传导抑制剂。我们在此描述该测定的开发和性能，并讨论其特定功能的优点。
A cell-sensor assay for stabilization of I kappa B alpha was developed in the activated B cell-like diffuse large B-cell lymphoma cell line OCI-Ly3. This cell line expresses known nuclear factor kappa B (NF kappa B) target genes due to high constitutive activity of I kappa B kinase (IKK), which phosphorylates the protein I kappa B alpha leading to proteasomal degradation of I kappa B alpha and activation of NF kappa B. The cell-sensor assay uses green and red light-emitting beetle luciferases, with the green luciferase fused to I kappa B alpha (I kappa B alpha-CBG68) and the red luciferase (CBR) present in its native state. The I kappa B alpha-CBG68 reporter functions as a sensor of IKK and proteasome activity, while CBR serves to normalize for cell number and nonspecific effects. Both reporter constructs were stably integrated and placed under the control of an inducible promoter system, which increased fold responsiveness to inhibitors when assay incubations were performed simultaneous to reporter induction by doxycycline. The assay was miniaturized to a 1,536-well plate format and showed a Z' of 0.6; it was then used to panel 2,677 bioactive compounds by a concentration-response-based screening strategy. The concentration-effect curves for the I kappa B alpha-CBG68 and CBR signals were then used to identify specific stabilizers of I kappa B alpha, such as IKK inhibitors or proteasome inhibitors, which increased the doxycycline-induced rise in I kappa B alpha-CBG68 without affecting the rise in CBR. Known and unexpected inhibitors of NF kappa B signaling were identified from the bioactive collection. We describe here the development and performance of this assay, and discuss the merits of its specific features.